Construction and Bioinformatics Analysis of ceRNA Regulatory Networks in Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive form of pulmonary fibrosis of unknown etiology. Despite ongoing research, there is currently no cure for this disease. Recent studies have highlighted the significance of competitive endogenous RNA (ceRNA) regulatory networks in IPF development. Therefore, this study investigated the ceRNA network associated with IPF pathogenesis. We obtained gene expression datasets (GSE32538, GSE32537, GSE47460, and GSE24206) from the Gene Expression Omnibus (GEO) database and analyzed them using bioinformatics tools to identify differentially expressed messenger RNAs (DEmRNAs), microRNAs (DEmiRNAs), and long non-coding RNAs (DElncRNA). For DEmRNAs, we conducted an enrichment analysis, constructed protein-protein interaction networks, and identified hub genes. Additionally, we predicted the target genes of differentially expressed mRNAs and their interacting long non-coding RNAs using various databases. Subsequently, we screened RNA molecules with ceRNA regulatory relations in the lncACTdb database based on the screening results. Furthermore, we performed disease and functional enrichment analyses and pathway prediction for miRNAs in the ceRNA network. We also validated the expression levels of candidate DEmRNAs through quantitative real-time reverse transcriptase polymerase chain reaction and analyzed the correlation between the expression of these candidate DEmRNAs and the percent predicted pre-bronchodilator forced vital capacity [%predicted FVC (pre-bd)]. We found that three ceRNA regulatory axes, specifically KCNQ1OT1/XIST/NEAT1-miR-20a-5p-ITGB8, XIST-miR-146b-5p/miR-31-5p- MMP16, and NEAT1-miR-31-5p-MMP16, have the potential to significantly affect IPF progression. Further examination of the underlying regulatory mechanisms within this network enhances our understanding of IPF pathogenesis and may aid in the identification of diagnostic biomarkers and therapeutic targets.

A retrospective study of ovarian tissue cryopreservation in female patients with hematological diseases for fertility preservation.

PURPOSES: To investigate the effect and safety of ovarian tissue cryopreservation (OTC) for fertility preservation in female patients with hematological diseases. METHODS: We designed a retrospective study. The clinical data of patients with hematological diseases undergoing OTC admitted to Peking University People's Hospital from April 2017 to January 2023 were analyzed and summarized. RESULTS: A total of 24 patients were included in the study, including 19 patients with malignant hematological diseases and 5 patients with non-malignant hematological diseases. The former included 14 patients with acute leukemia, 1 patient with chronic leukemia, and 4 patients with myelodysplastic syndrome, while the latter 5 patients were aplastic anemia (AA). 16 patients had received chemotherapy before OTC. The average age of 24 patients was 22.80 ± 6.81 years. The average anti-Mullerian hormone (AMH) was 1.97 ± 2.12 ng/mL, and the average follicle-stimulating hormone (FSH) was 7.01 ± 4.24 IU/L in examination before OTC. FSH was greater than 10.0 IU/L in 4 cases. The pre-OTC laboratory tests showed that the average white blood cell (WBC) count was (3.33 ± 1.35) × 109/L, the average hemoglobin was 91.42 ± 22.84 g/L, and the average platelet was (147.38 ± 114.46) × 109/L. After injection of recombinant human granulocyte colony-stimulating factor (rhG-CSF), blood transfusion, and iron supplementation in pre-OTC treatment, the average WBC count was (4.91 ± 3.07) × 109/L, the average hemoglobin was 98.67 ± 15.43 g/L, and the average platelet was (156.38 ± 103.22) × 109/L. Of the 24 patients, 22 underwent laparoscopic bilateral partial oophorectomy and oophoroplasty, and 2 underwent laparoscopic unilateral oophorectomy. The average duration of OTC was 59.54 ± 17.58 min, and the average blood loss was 32.1 ± 41.6 mL. The maximum blood loss was 200 mL. There was no significant difference in WBC count and hemoglobin concentration after OTC compared to pre-OTC period. Only the platelet count after OTC surgery was significantly different from that before surgery ([134.54 ± 80.84 vs. 156.38 ± 103.22] × 109/L, p < 0.05). None of the 24 patients had serious complications after OTC. 2 patients had mild infection symptoms, but both recovered well. 23 patients underwent hematopoietic stem cell transplantation (HSCT) after OTC. The median and interquartile range from OTC to the pretreatment of HSCT was 33 (57) days, and the median and interquartile range from OTC to HSCT was 41 (57) days. Seven of them began pretreatment of HSCT within 20 days and began HSCT within 30 days after OTC. All patients were followed up. Of the 23 patients who underwent HSCT after surgery, 22 presented with amenorrhea and 1 with scanty menstrual episodes. Seven patients underwent hormone replacement therapy (HRT) after HSCT. A patient with AA underwent ovarian tissue transplantation (OTT) 3 years after HSCT and resumed regular menstruation 6 months after OTT. CONCLUSIONS: Ovarian tissue cryopreservation has a promising future in fertility protection in patients with hematological diseases. However, patients with hematological malignancies often have received gonadotoxic therapy before OTC, which may be accompanied by myelosuppression while patients with non-malignant hematological diseases often present with severe hemocytopenia. So perioperative complete blood count of patients should be paid attention to. There was no significant difference in the WBC count and hemoglobin concentration in patients with hematological diseases before and after OTC surgery, and the platelet count decreased slightly within the normal range. Infection is the most common post-OTC complication, and HSCT pretreatment can be accepted as early as the 10th day after OTC. OTC has no adverse effects on patients with hematological diseases and does not delay HSCT treatment. For young patients with hematological diseases, OTC is an effective method of fertility preservation.

PRKDC-Mediated NHEJ May Play a Crucial Role in Aneuploidy of Chromosome 8-Driven Progression of Ovarian Cancer.

High malignancy is a prominent characteristic of epithelial ovarian cancer (EOC), emphasizing the necessity for further elucidation of the potential mechanisms underlying cancer progression. Aneuploidy and copy number variation (CNV) partially contribute to the heightened malignancy observed in EOC; however, the precise features of aneuploidy and their underlying molecular patterns, as well as the relationship between CNV and aneuploidy in EOC, remain unclear. In this study, we employed single-cell sequencing data along with The Cancer Genome Atlas (TCGA) to investigate aneuploidy and CNV in EOC. The technique of fluorescence in situ hybridization (FISH) was employed using specific probes. The copy number variation within the genomic region of chromosome 8 (42754568-47889815) was assessed and utilized as a representative measure for the ploidy status of individual cells in chromosome 8. Differential expression analysis was performed between different subgroups based on chromosome 8 ploidy. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), protein-protein interaction (PPI), and hub-gene analyses were subsequently utilized to identify crucial genes involved. By classifying enriched tumor cells into distinct subtypes based on chromosome 8 ploidy combined with TCGA data integration, we identified key genes driving chromosome 8 aneuploidy in EOC, revealing that PRKDC gene involvement through the mediated non-homologous end-joining pathway may play a pivotal role in disease progression. Further validation through analysis of the GEO and TCGA database and survival assessment, considering both mRNA expression levels and CNV status of PRKDC, has confirmed its involvement in the progression of EOC. Further functional analysis revealed an upregulation of PRKDC in both ovarian EOC cells and tissues, with its expression showing a significant correlation with the extent of copy number variation (CNV) on chromosome 8. Taken together, CNV amplification and aneuploidy of chromosome 8 are important characteristics of EOC. PRKDC and the mediated NHEJ pathway may play a crucial role in driving aneuploidy on chromosome 8 during the progression of EOC.

Autologous non-invasively derived stem cells mitochondria transfer shows therapeutic advantages in human embryo quality rescue.

BACKGROUND: The decline in the quantity and quality of mitochondria are closely associated with infertility, particularly in advanced maternal age. Transferring autologous mitochondria into the oocytes of infertile females represents an innovative and viable strategy for treating infertility, with no concerns regarding ethical considerations. As the donor cells of mitochondria, stem cells have biological advantages but research and evidence in this area are quite scarce. METHODS: To screen out suitable human autologous ooplasmic mitochondrial donor cells, we performed comprehensive assessment of mitochondrial physiology, function and metabolic capacity on a varity of autologous adipose, marrow, and urine-derived mesenchymal stromal cells (ADSC, BMSC and USC) and ovarian germline granulosa cells (GC). Further, to explore the biosafety, effect and mechanism of stem cell-derived mitochondria transfer on human early embryo development, randomized in-vitro basic studies were performed in both of the young and aged oocytes from infertile females. RESULTS: Compared with other types of mesenchymal stromal cells, USC demonstrated a non-fused spherical mitochondrial morphology and low oxidative stress status which resembled the oocyte stage. Moreover, USC mitochondrial content, activity and function were all higher than other cell types and less affected by age, and it also exhibited a biphasic metabolic pattern similar to the pre-implantation stage of embryonic development. After the biosafety identification of the USC mitochondrial genome, early embryos after USC mitochondrial transfer showed improvements in mitochondrial content, activity, and cytoplasmic Ca2+ levels. Further, aging embryos also showed improvements in embryonic morphological indicators, euploidy rates, and oxidative stress status. CONCLUSION: Autologous non-invasively derived USC mitochondria transfer may be an effective strategy to improve embryonic development and metabolism, especially in infertile females with advanced age or repeated pregnancy failure. It provides evidence and possibility for the autologous treatment of infertile females without invasive and ethical concerns.

A selective CB2R agonist (JWH133) protects against pulmonary fibrosis through inhibiting FAK/ERK/S100A4 signaling pathways.

BACKGROUND: The combination of the endocannabinoid system (ECS) and the type 2 cannabinoid receptor (CB2R) can activate various signal pathways, leading to distinct pathophysiological roles. This interaction has gained significant attention in recent research on fibrosis diseases. Focal adhesion kinase (FAK) plays a crucial role in regulating signals from growth factor receptors and Integrins. It is also involved in the transformation of fibroblasts into myofibroblasts. This study aims to investigate the impact of the CB2R agonist JWH133 on lung fibrosis and its potential to alleviate pulmonary fibrosis in mice through the FAK pathway. METHODS: The C57 mice were categorized into five groups: control, BLM, BLM + JWH133, BLM + JWH133 + NC, and BLM + JWH133 + FAK groups.JWH133 was administered to mice individually or in conjunction with the FAK vector. After 21 days, pathological changes in mouse lung tissues, inflammatory factor levels, hydroxyproline levels, and collagen contents were evaluated. Moreover, the levels of the FAK/ERK/S100A4 pathway-related proteins were measured. RESULTS: JWH133 treatment decreased inflammatory factor levels, attenuated pathological changes, and reduced extracellular matrix accumulation in the mouse model of bleomycin-induced pulmonary fibrosis; however, these effects were reversed by FAK. JWH133 attenuated fibrosis by regulating the FAK/ERK/S100A4 pathway. CONCLUSIONS: The results presented in this study show that JWH133 exerts a protective effect against pulmonary fibrosis by inhibiting the FAK/ERK/S100A4 pathway.Therefore, JWH133 holds promise as a potential therapeutic target for pulmonary fibrosis.

Accuracy of transvaginal ultrasound and magnetic resonance imaging for diagnosis of deep endometriosis in bladder and ureter: a meta-analysis.

This meta-analysis aimed to determine the accuracy of transvaginal ultrasound (TVS) and pelvic magnetic resonance imaging (MRI) in diagnosing urinary tract endometriosis (UTE). A comprehensive search of the Pubmed and Embase was conducted between January 1989 and June 2020. Studies that described the accuracy of MRI or TVS for the diagnosis of UTE using surgical data as the reference standard were included. Of the 913 citations identified, 23 studies were analysed. For detection of endometriosis in bladder endometriosis (BE), the overall pooled sensitivities of TVS and MRI were 72% and 68% respectively, and their specificities were 99% and 100% respectively. For detection of endometriosis in the ureteral endometriosis (UE), the overall pooled sensitivities of TVS and MRI were 97% and 87% respectively, and their specificities were both 100%. In conclusion, both TVS and MRI provide good accuracy with specific strong points in diagnosing UTE and seem useful first-line methods from a clinical perspective. Besides, pelvic MRI and TVS are more accurate for predicting UTE localised in the ureter than bladder, especially in terms of sensitivity.IMPACT STATEMENTWhat is already known on this subject? Previous studies have confirmed high diagnostic value of transvaginal ultrasound (TVS) and magnetic resonance imaging (MRI) on bladder endometriosis (BE) respectively. However, high heterogeneity was found for both sensitivity and specificity and no meta-analysis has yet been performed to test the diagnostic value of TVS and MRI for ureteral endometriosis (UE).What the results of this study add? In this meta-analysis, we firstly confirmed high diagnostic value of TVS and MRI on UE respectively. For detection of UE, the overall pooled sensitivities of TVS and MRI were 97% and 87% respectively, and their specificities were both 100%.What the implications are of these findings for clinical practice and/or further research? Early preoperative diagnosis and accurate understanding of the widespread distribution of endometriosis are prerequisites for radical surgical in UTE. In the present study, we updated the previous results on the accuracy of TVS and MRI for the diagnosis of BE and firstly confirmed high diagnostic value of TVS and MRI on UE. Both TVS and MRI provide good accuracy with specific strong points in diagnosing UTE and seem useful first-line methods from a clinical perspective.

Antral follicle count is reduced in the presence of endometriosis: a systematic review and meta-analysis.

Studies suggest that the presence of endometriosis may lead to impaired ovarian reserve, while results evaluating the changes in antral follicle count (AFC) in endometriosis remain controversial. A systematic search returned 15 studies, of which nine compared AFC between patients with and without endometriosis, five articles reported differences in AFC between affected and unaffected ovaries in patients with unilateral ovarian endometriosis and one reported both of the above two situations. Overall results showed a significant decrease in AFC and anti-Müllerian hormone (AMH) and increase in serum FSH concentrations in patients with endometriosis when compared with controls. Additionally, the AFC for the ovary with the endometrioma was also significantly lower than that of the contralateral ovary in patients with unilateral ovarian endometriosis. Moreover, it appears that the AFC in patients with endometriosis where the ovaries are not affected or in early stage were not significantly different in the control group. These findings demonstrate that endometriosis is associated with reduced AFC and AMH and elevated serum concentrations of FSH, suggesting a reduction in ovarian reserve in patients with endometriosis, especially in those with ovarian endometrioma and advanced stage.

Clinical characteristics, treatment status and complications in women with tube ovarian abscess and endometriosis: a retrospective study.

BACKGROUND: The aim of our present study was to investigate the clinical characteristics, treatment status and complications in women with endometriosis (EM) and tube ovarian abscess (TOA) to determine the possible association between TOA and EM. METHODS: Medical records were used to analyze the clinical characteristics, treatment and complications. Twenty women who were diagnosed with TOA with EM were compared with 93 women diagnosed as having TOA without EM between January, 2008 and December, 2018. RESULTS: In this study, TOA patients with EM were significantly more likely to have a lower age range (20-39 years) than the non-EM group [11/20 (55.0%) vs 27/93 (29.0%)]. In addition, TOA patients with EM were associated with a significantly lower rate of parity [11/20 (55.0%) vs 75/93 (80.6%)], higher rates of infertility [8/20(40%) vs 0/93(0%)] and a significantly lower incidence of elevated blood platelet counts [5/20 (25%) vs 43/93 (46.2%)]. Furthermore, women with EM had greater blood loss (347 ± 445.77 vs 204.67 ± 289.46) and an increased complication rate [3/20(15%) vs 0/93(0%)]. Among the 3 patients who had complications in the EM group, 2 patients had septic shock and 1 patient had intestinal obstruction. And 1 case who had septic shock followed by IVF treatment. There was no significance difference on other factors. CONCLUSIONS: The present study indicated that EM did not increase the difficulty and time of treatment in patients with TOA, but increased bleeding during surgery and serious complications. It is suggested that doctors should pay more attention to postoperative treatment and nursing in women with TOA and EM, especially those who have a history of recent infertility treatment and related procedures.

Serum and peritoneal fluid leptin levels in endometriosis: a systematic review and meta-analysis.

The role of leptin in the development of endometriosis has been investigated previously. However, researches on the change of leptin levels in endometriosis remains controversial. So, we aimed to clarify changes of leptin levels in patients with endometriosis and their association with the progression of endometriosis. We searched PubMed, Embase, Web of Science, and The Cochrane Library to identify relevant studies published before May 25, 2020. The detected levels of leptin in patients with endometriosis versus controls were evaluated in this meta-analysis. Eighteen studies met our inclusion criteria, five studies detected serum, nine detected peritoneal fluid and another four detected both serum and peritoneal fluid leptin levels. The overall results showed that peritoneal fluid leptin levels in patients with endometriosis was significantly higher than that in the control group, but the serum and corrected peritoneal fluid leptin levels were comparable in both groups. Subgroup analysis failed to eliminate the high degree of heterogeneity included in the studies and showed that peritoneal fluid leptin levels were significantly elevated in both early and advanced endometriosis. In conclusion, peritoneal fluid rather than serum leptin levels was elevated in patients with endometriosis, which did not seem to be related to the severity of endometriosis, but was related to body mass index.

Combined detection and subclass characteristics analysis of CTCs and CTECs by SE-iFISH in ovarian cancer.

OBJECTIVE: Hematogenous metastasis is essential for the progression of ovarian cancer (OC), and circulating tumor cells (CTCs) are part of the metastatic cascade. However, the detection rate of CTC is low due to the use of less sensitive detection methods. Therefore, this study aimed to detect CTCs and circulating tumorigenic endothelial cells (CTECs) in patients with OC using subtraction enrichment and immunostaining and fluorescence in situ hybridization (SE-iFISH). METHODS: We enrolled a total of 56 subjects, including 20 OC patients and 36 ovarian benign tumor patients. CTCs and CTECs were captured by subtraction enrichment (SE) and counted and classified according to immunofluorescence staining of tumor markers (TMs) carbohydrate antigen 125 (CA125) and human epididymis protein 4 (HE4) combined with fluorescence in situ hybridization (iFISH) of chromosome 8 (Chr8) aneuploidy. The diagnostic value and subtype characteristics of CTCs and CTECs were investigated. RESULTS: The detection rate of CTCs by SE-iFISH was high. Compared with CA125 and HE4, Chr8 aneuploidy was the major identification feature of CTC. CTC counts in OC were statistically higher than those in benign groups. CTC and CTEC with ≥pentaploidy were detected in both groups, illustrating the poor diagnostic value of CTC or CTEC. Distributions of triploid and tetraploid CTC subtypes were significantly different, and combined detection of triploid and tetraploid CTCs showed the best diagnostic value. In contrast, the distribution of CTECs in the OC and benign groups had no statistically significant difference. Small CTCs accounted for over 1/3 of the total CTC count. We also found that small CTCs and CTECs primarily comprised triploid cells, while large CTCs and CTECs mainly comprised pentaploidy and beyond. CONCLUSIONS: The application of SE-iFISH offered a more comprehensive understanding of heterogeneous CTCs and CTECs in OC. Analysis of subclass characteristics of the CTCs and CTECs according to Chr8 aneuploidy and cell size may broaden their potential clinical utility and deepen mechanistic studies in OC.

Fibrinogen alpha chain promotes the migration and invasion of human endometrial stromal cells in endometriosis through focal adhesion kinase/protein kinase B/matrix metallopeptidase 2 pathway†.

Fibrinogen alpha chain (FGA), a cell adhesion molecule, contains two arginyl-glycyl-aspartic acid (RGD) cell adhesion sequences. Our previous study demonstrated that FGA, as an up-regulated protein in endometriosis (EM), was closely related to disease severity and involved in the development of EM. However, the biological functions and underlying mechanism of FGA in EM have not been fully understood. To explore the roles of FGA in EM, we analyzed the effects of FGA on the biological behaviors of human primary eutopic endometrial stromal cells (EuESC). The results indicated FGA knockdown suppressed the migration and invasion ability of EuESC, which also altered the distribution of cytoskeletal filamentous and cell morphology. Western blot analysis demonstrated that knockdown of FGA attenuated the migration-related protein levels of vimentin and matrix metallopeptidase 2 (MMP-2), but not integrin subunit alpha V (ITGAV) and integrin subunit beta 3 (ITGB3). Meanwhile, integrin-linked transduction pathways were detected. We found FGA knockdown significantly suppressed the expression of focal adhesion kinase (FAK) level and protein kinase B (AKT) phosphorylation, without extracellular-signal-regulated kinase (ERK) dependent pathways. Treatment with the AKT inhibitor MK2206 or RGD antagonist highly decreased the effects of FGA on the migration and invasion of EuESC. RGD antagonist treatment strongly inhibited FAK- and AKT-dependent pathways, but not ERK pathways. Our data indicated that FGA may enhance the migration and invasion of EuESC through RGD sequences binding integrin and activating the FAK/AKT/MMP-2 signaling pathway. This novel finding suggests that FGA may provide a novel potential approach to the treatment of EM, which provides a new way to understand the pathogenesis of EM.

IL-32 induces epithelial-mesenchymal transition by triggering endoplasmic reticulum stress in A549 cells.

BACKGROUND: Epithelial-mesenchymal transition (EMT) is a key process in the onset and development of idiopathic pulmonary fibrosis (IPF) with unclear mechanisms. Our previous studies found that bleomycin and tunicamycin could induce ER stress and consequently trigger EMT accompanying with IL-32 overexpression. This study was aimed to investigate the effects of IL-32 on EMT and ER stress to elucidate the pathogenesis of IPF. METHODS: Human lung adenocarcinoma A549 cells were treated with recombinant human (rh)IL-32, IL-32 siRNA and EMT inducer tunicamycin, or 4-phenylbutyric acid (4-PBA), respectively. Then the cell morphology was observed and the expression of ER-related markers and EMT-related markers were detected by RT-qPCR or western blotting. RESULTS: Stimulation of A549 cells with rhIL-32 led to a morphological change from a pebble-like shape to an elongated shape in a portion of the cells, accompanied by down regulated expression of the epithelial cell marker E-cadherin and up regulated expression of the mesenchymal cell markers N-cadherin, Vimentin, and Zeb-1. However, these rhIL-32 induced changes were inhibited by the ER stress inhibitor 4-PBA. Suppression of IL-32 expression with siRNA inhibited TM-induced EMT. Further stimulation of the A549 cells with rhIL-32 demonstrated an increase in the expression of GRP78, although this increase was also inhibited by 4-PBA. CONCLUSIONS: These results suggest that IL-32 induces EMT in A549 cells by triggering ER stress, and IL-32 may be a novel marker for IPF.

Knockdown of long noncoding RNA CCDC144NL-AS1 attenuates migration and invasion phenotypes in endometrial stromal cells from endometriosis†.

Endometriosis (EM) is a mysterious and complicated disease that has been found to be multifactorial. Recent studies demonstrated that long noncoding RNAs (lncRNAs) play an important role in the pathogenesis of EM. However, the functional and biological mechanisms of lncRNAs in EM remain unknown. Here, we performed microarray analyses to compare the lncRNA expression profiles of four paired ectopic endometrial (EC) tissues and eutopic endometrial (EU) tissues from patients with ovarian EM. A novel lncRNA, CCDC144NL-AS1, was identified as being potentially functional. CCDC144NL-AS1 expression was upregulated in EC tissues compared to EU and normal endometrial (NE) tissues. Its expression was higher in EC tissues than in EU tissues in 86.7% (26/30) of patients with EM. Despite the lack of a significant increase according to revised American Fertility Society (rAFS) stages, approximately 60% of stage VI EM cases exhibited higher CCDC144NL-AS1 levels, many more than in the stage II-III cases. Subcellular fractionation demonstrated that CCDC144NL-AS1 was localized in the cytoplasm and nucleus of the human EM-derived immortalized endometrial stromal cell line hEM15A. CCDC144NL-AS1 depletion suppressed the migration and invasion of hEM15A cells, but exerted no effects on cell adhesion, proliferation, apoptosis, or cell cycle. Knockdown of CCDC144NL-AS1 dramatically altered the distribution of cytoskeletal filamentous actin (F-actin) stress fibers compared to the negative control group treatment. Western blot analysis revealed that knockdown of CCDC144NL-AS1 attenuated the protein levels of vimentin filaments and MMP-9, but not N-cadherin or ß-catenin. Collectively, our results suggest that CCDC144NL-AS1 might be involved in the pathogenesis of EM and provide a novel target for ovarian EM.

Histone Deacetylases Promote ER Stress Induced Epithelial Mesenchymal Transition in Human Lung Epithelial Cells.

BACKGROUND/AIMS: Epithelial to mesenchymal transition (EMT) is a crucial process involved in pulmonary fibrosis. This study aimed to explore the role of histone deacetylases (HDACs) and endoplasmic reticulum (ER) stress in EMT in human lung epithelial cells. METHODS: Human lung adenocarcinoma A549 cells were treated with bleomycin and tunicamycin to induce EMT. The proliferation of A549 cells was detected by MTT assay. The expression of HDACs and EMT markers was detected by PCR and Western blot analysis. The secretion of TGF-ß1 and collagen I was examined by ELISA. RESULTS: A549 cells switched from a cobblestone-like appearance to an elongated fibroblast like appearance after exposure to tunicamycin or bleomycin, accompanied by increased expression of N-cadherin, α-SMA and Collagen I. Meanwhile, GRP78 was upregulated in A549 cells exposed to tunicamycin or bleomycin. These changes induced by tunicamycin or bleomycin could be abrogated by 4-PBA. Moreover, tunicamycin and bleomycin promoted the expression of HDAC2 and HDAC6, and HDACs inhibitor SAHA abrogated the morphological and biochemical changes in A549 cells. 4-PBA and SAHA inhibited the upregulation of pulmonary fibrosis factors TGF-ß1 and IL-32 and the activation of Smad pathway induced by tunicamycin or bleomycin. CONCLUSIONS: We provide the first evidence that tunicamycin and bleomycin induce ER stress and EMT in lung epithelial cells via the upregulation of HDACs. HDACs inhibitor could inhibit ER stress induced upregulation of pulmonary fibrosis factors and the activation of Smad pathway. HDACs inhibitors are promising agents for the therapy of pulmonary fibrosis.

[Effect of Curcumin on TGF-ß2 Regulated PPAR-γ/PDGF-ß Signaling Pathway in Lung Fibroblasts of Mice].

OBJECTIVE: To explore the effect of curcumin on TGF-ß2 regulated peroxisome proliferater activated receptor y (PPAR-γ)/platelet derived growth factor ß (PDGF-ß) signaling pathway in lung fibroblasts of mice. METHODS: C57BL/6 mouse lung fibroblasts were in vitro cultured with TGF-ß2, curcumin, or TGF-ß2 plus curcumin. The cell proliferation was detected by cell growth counting in the blank control group, low, middle, and high dose curcumin groups (5, 25, 50 µmol/L), the TGF-ß2 (10 ng/mL) group, TGF-ß2 (10 ng/mL) plus curcumin (5, 25, 50 µmol/L) groups. mRNA expressions of PPAR-γ, platelet-derived growth factor receptor ß (PDGFR-ß), fibroblast growth factor R1 (FGFR1) were detected using reverse transcription PCR. Protein levels of PPAR-γ and collagen-1 were detected using Western blot and ELISA in the blank control group, the TGF-ß2 group, the TGF-ß2 (10 ng/mL) plus curcumin 50 µmol/L group. RESULTS: Compared with the blank control group, curcumin 50 µmol/L showed the most significant inhibition on cell proliferation at 48 h and 72 h. Compared with the TGF-ß2 group, TGF-ß2 (10 ng/mL) plus curcumin 50 mol/L also showed the most significant inhibition on cell proliferation at 48 h and 72 h. Compared with the blank control group, mRNA expressions of PPAR-γ and PDGF-ß, as well as protein expression of PPAR-γ increased, the collagen-1 expression also increased in the TGF-ß2 group (P < 0.05). Compared with the TGF-ß2 group, mRNA expressions of PPAR-γ obviously increased in the TGF-ß2 (10 ng/mL) plus curcumin 25 µmol/L group and the TGF-ß2 (10 ng/mL) plus curcumin 50 µmol/L group, higher than that in the TGF-ß2 (10 ng/mL) plus curcumin 5 [µmol/L group (P < 0.05). mRNA expressions of PPAR-γ was higher in the TGF-ß2 (10 ng/mL) plus curcumin 50 µmol/L group than in the TGF-ß2 (10 ng/mL) plus curcumin 25 µmol/L group (P < 0.05). mRNA expressions of PDGF-ß was lower in TGF-ß2 (10 ng/mL) plus curcumin groups than in the TGF-ß2 group (P < 0.05). Besides, PDGF-ß mRNA expressions were lower in the TGF-ß2 (10 ng/mL) plus curcumin 50 µmol/L group than in the TGF-ß2 (10 ng/mL) plus curcumin 5 µmol/L group and the TGF-ß2 (10 ng/mL) plus curcumin 25 µmol/L group (P < 0.05). There was no statistical difference in FGFR1 mRNA expressions between the TGF-ß2 group and 3 TGF-ß2 plus curcumin groups (P > 0.05). Compared with the TGF-ß2 group, PPAR-γ protein expressions increased and collagen-1 protein expressions decreased in the TGF-ß2 (10 ng/mL) plus curcumin 50 µLmol/L group (P < 0.05, P < 0.01). CONCLUSIONS: Curcumin not only could inhibit TGF-ß2 induced proliferation of lung fibroblasts, but also could inhibit the synthesis of collagens. These might be associated with up-regulating PPAR-γ expressions and down-regulating PDGF-ß expressions. Therefore, curcumin might inhibit the occurrence and developing of lung fibrosis through blocking PPAR-γ/PDGF-ß signaling pathway.

Precise capture of circulating endometrial cells in endometriosis.

BACKGROUND: Endometriosis (EM) is a complex benign gynecological disease, but it has malignant biological behavior and can invade any part of the body. Clinical manifestations include pelvic pain, dysmenorrhea, infertility, pelvic nodules, and masses. Our previous study successfully detected circulating endometrial cells (CECs) in the peripheral blood of patients with EM. The purpose of this study is to overcome the limitation of cell size in the previous microfluidic chip method, to further accurately capture CECs, understand the characteristics of these cells, and explore the relationship between CECs and the clinical course characteristics of patients with EM. METHODS: Human peripheral venous blood used to detect CECs and circulating vascular endothelial cells (CVECs) was taken from EM patients (n = 34) hospitalized in the Peking University People's Hospital. We use the subtraction enrichment and immunostaining fluorescence in situ hybridization (SE-iFISH) method to exclude the interference of red blood cells, white blood cells, and CVECs, so as to accurately capture the CECs in the peripheral blood of patients with EM. Then we clarify the size and ploidy number of chromosome 8 of CECs, and a second grouping of patients was performed based on clinical characteristics to determine the relationship between CECs and clinical course characteristics. RESULTS: The peripheral blood of 34 EM patients and 12 non-EM patients was evaluated by SE-iFISH. Overall, 34 eligible EM patients were enrolled. The results showed that the detection rates of CECs were 58.8% in EM patients and 16.7% in the control group. However, after classification according to clinical characteristics, more CECs could be detected in the peripheral blood of patients with rapidly progressive EM, with a detection rate of 94.4% (17/18). In total, 63.5% (40/63) of these cells were small cells with diameters below 5 µm, and 44.4% (28/63) were aneuploid cells. No significant association was found between the number of CECs and EM stage. CONCLUSION: The number and characteristics of CECs are related to the clinical course characteristics of patients with EM, such as pain and changes in lesion size, and may be used as biomarkers for personalized treatment and management of EM in the future.

ECM1 promotes migration and invasion in endometriosis.

Extracellular matrix protein 1 (ECM1) is a glycoprotein that may be a key player in tumorigenesis and tumor progression. However, knowledge regarding the role of ECM1 in endometriosis (EM) is still lacking. Microarray analyses were performed to compare the mRNA expression patterns between paired EU tissues and ectopic endometrial (EC) tissues (n = 4) from EM patients. ECM1 expression was significantly increased in the eutopic endometrial (EU) tissues than paired EC tissues of endometriotic patients and normal endometrial (NE) tissues of controls without EM. Blocking ECM1 with siRNA attenuated the migration and invasion of hEM15A cells and modified the distribution of the F-actin cytoskeleton. We conducted microarray analyses and bioinformatics analyses to investigate the differentially expressed genes (DEGs) and related pathways regulated by ECM1. A total of 161 DEGs between the siECM1 and the negative control (siNC) treatments were identified, consisting of 79 downregulated genes and 82 upregulated genes. Enriched DEGs were associated with 9 gene ontology (GO) terms. Moreover, a protein-protein interaction (PPI) network was constructed for the hub genes and modules. Radixin (RDX) was the second most downregulated gene in the siECM1 group compared with the siNC group. ECM1 knockdown significantly decreased the expression of RDX, RhoC, ROCK1, N-cadherin and ß-catenin but not ROCK2. ECM1 showed high tissue-specific expression in EU tissues from EM patients, and may contribute to the migration, invasion and reorganization of the F-actin cytoskeleton in eutopic endometrial stromal cells via the RhoC/ROCK1 signaling pathway in EM.

Postmenopausal endometrial non-benign lesion risk classification through a clinical parameter-based machine learning model.

OBJECTIVE: This study aimed to develop and evaluate a machine learning model utilizing non-invasive clinical parameters for the classification of endometrial non-benign lesions, specifically atypical hyperplasia (AH) and endometrioid carcinoma (EC), in postmenopausal women. METHODS: Our study collected clinical parameters from a cohort of 999 patients with postmenopausal endometrial lesions and conducted preprocessing to identify 57 relevant characteristics from these irregular clinical data. To predict the presence of postmenopausal endometrial non-benign lesions, including atypical hyperplasia and endometrial cancer, we employed various models such as eXtreme Gradient Boosting (XGBoost), Random Forest (RF), Logistic Regression (LR), Support Vector Machine (SVM), Back Propagation Neural Network (BPNN), as well as two ensemble models. Additionally, a test set was performed on an independent dataset consisting of 152 patients. The performance evaluation of all models was based on metrics including the area under the receiver operating characteristic curve (AUC), sensitivity, specificity, precision, and F1 score. RESULTS: The RF model demonstrated superior recognition capabilities for patients with non-benign lesions compared to other models. In the test set, it attained a sensitivity of 88.1% and an AUC of 0.93, surpassing all alternative models evaluated in this study. Furthermore, we have integrated this model into our hospital's Clinical Decision Support System (CDSS) and implemented it within the outpatient electronic medical record system to continuously validate and optimize its performance. CONCLUSIONS: We have trained a model and deployed a system with high discriminatory power that may provide a novel approach to identify patients at higher risk of postmenopausal endometrial non-benign lesions who may benefit from more tailored screening and clinical intervention.